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Non-clinical Drug Toxicokinetics Research

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3.1. General principles of non-clinical toxicology research

Toxicokinetics (TK) is a cross-discipline that combines pharmacokinetics (PK) and toxicology. It has now become an important part of drug development and non-clinical research. Toxicokinetics is the principle and method of applying pharmacokinetics. Quantitatively study the process and characteristics of drug absorption, distribution, metabolism, and excretion in animals at toxic doses, and a longer discussion on the occurrence and development of drug toxicity, understanding the distribution of drugs in animals and their target organs, for further Conduct other toxicity tests and clinical trials to provide a basis for future clinical drug use, as well as the diagnosis and treatment of drug overdose.

drug toxicokinetics

Under toxic doses, the interaction between the transport system and the kinetic mechanism of the toxic reaction of metabolism in the body and pharmacokinetics is that the doses for the absorption, distribution, metabolism and excretion of the researched drug are much higher than the usual doses for drug screening or drug treatment . Therefore, toxicokinetics is not a simple description of the basic kinetic characteristics or toxic reaction events of the test substance. Rather, the enzyme may become saturated, the protein binding rate may change, and the overall response of the physiological system may also change. . To establish concentration-response relationships and concentration-effect relationships more scientifically. The purpose of toxicokinetic studies is to know the degree and duration of systemic exposure of the test substance at different dose levels in the toxicity test, and to predict the test substance’s exposure to humans. Potential risks, the focus of research is to interpret the results of toxicity tests and predict human safety.

For innovative drugs, toxicokinetic research has obvious value in the following aspects:

    Describe the relationship between the systemic exposure and toxicity of the test substance in the toxicity test;

    Describe the effects of repeated drug exposure and prolonged exposure on metabolic processes, including effects on metabolic enzymes (such as the induction or inhibition of drug-metabolizing enzymes);

    Explain the toxicological findings or changes of the test substance in the toxicity test,

    To evaluate the toxicity of the test substance in different animal species, gender, age, and body conditions (such as disease or pregnancy status);

    Animal species selection and medication plan to support non-clinical toxicity studies;

    Analyzing the predictive value of animal toxicity for clinical safety evaluation, such as liver toxicity or kidney damage caused by drug accumulation, can provide quantitative information for subsequent safety evaluation;

    Comprehensive drug efficacy and exposure, toxicity and exposure information to guide the design of human trials, such as initial dose, safety range evaluation, etc., and guide clinical safety monitoring according to the degree of exposure;

    In some cases, short-term toxicity tests (1 to 3 months) accompanied by toxicokinetic studies can better support the entry of drugs into early clinical trials, thereby reducing the safety risks of clinical trials and the development cycle of alternative drugs.

3.2. Physicochemical and metabolic characteristics of biotechnology drugs

Compared with small molecule chemical drug labeling, biotechnology drugs are significantly different from the physical and chemical properties of the drug molecule itself. There are some differences in the characteristics of RK and TK, and there are some particularities. For example, the potential immunogenicity of macromolecular protein drugs may affect TK behavior and TK parameters, thereby affecting efficacy and toxicity.

3.3. The main parameters of toxicokinetics

The basic purpose of the toxicokinetic test is to assess the systemic exposure of the test substance (and/metabolite, for small molecule drugs). The degree of exposure can be expressed by the plasma (serum or whole blood) concentration or AUC of the prototype test substance. The toxicokinetic study calculates the kinetic parameters by determining the sample concentration at the appropriate time point. The following content will briefly introduce how to use the PK principle for TK evaluation.

Pre-clinical PK and TK studies generally choose non-compartmental models (NCA) to fit drug-time data and calculate kinetic parameters. Because NCA’s earlier compartment model is flexible and durable, it is most commonly used in preclinical drug research.

The basis of the toxicity test should be the same as the clinical approach, so small molecules are mostly oral; biotech drugs are mostly intravenous (IVV) due to their poor oral bioavailability. The duration of the process is divided into intravenous bolus injection and intravenous slow injection. The former is completed within 1 minute; approximately 1 to 2 minutes, but not more than 15 minutes intravenous access. Because there is no absorption process, the correct clearance rate distribution can be obtained. Volume and biological half-life

1. Clearance rate

The clearance rate is often expressed as “CL”, also known as the total body clearance rate. The clearance rate is the volume of drug-containing plasma eliminated from the body per unit time or the apparent distribution space of the drug eliminated from the body per unit time.

2. Biological half-life

Biological half-life refers to the time required for the amount of drug in the body or blood drug concentration to be eliminated by various means. Biological half-life is an indicator of how quickly a drug is eliminated from the body.

3. In vivo exposure parameters Cm and AUC

Cm is the highest concentration measured in the biochip. AUC is the area under the plasma drug concentration-time curve. In the TK study, a comparison of exposures between different doses was used to calculate the accumulation ratio of multiple repetitions (especially drugs with long half-lives).

3.4. General principles of toxicokinetics research

1. Guiding principles and requirements

The ICH International Coordination Meeting on the Registration of Technical Requirements for Drugs for Human Use issued on October 27, 1994 the ICH tripartite (European Union, Japan, and the United States) coordinated guidelines-guidance on systemic exposure assessment in toxicity studies, and in 1995 It was published in the Federal Register of the US FDA in March (60FR11264), which is suitable for the toxicokinetic research of chemical drugs and biotechnology drugs.

The OECD issued a draft of the toxicokinetic guidelines (OECD Chemical Testing Guidelines: Toxicokinetics) in 2008, which came into effect on July 22, 2010.

It is expected that the “Technical Guidelines for Pharmacokinetics Research” will be released on May 13, 2014, which mainly refers to the format and content of ICH S3A.

Toxicity test studies are conducted in research institutions that comply with the Good Laboratory Practice (GLP) regulations for drug non-clinical safety research; sample analysis and data processing for toxicokinetic studies must comply with technical guidelines and Must comply with the principles of GLP.

2. Test animal selection, test substance, introduction method

In the TK study, the selection of animals, the test substance, and the method requirements are basically consistent with the PK study.

3. Dose selection

In principle, the repeated design of the TK test should be at least three dose groups, low, medium, and high, and one high dose. In principle, the animal will be fully exposed to produce obvious toxicity, or reach the maximum volume (MFD), or reach the system exposure The low dose of 50 times the clinical system exposure (based on AUC) is in principle equivalent to or higher than the equivalent dose of the animal pharmacodynamic dose or the clinically used dose. The middle dose should be established between the high dose and the low dose to check the dose-response relationship of toxicity. If it is necessary to change the dose in the middle of the test, the reason for the dose adjustment should be explained and the dose adjustment process should be fully recorded.

4. Sample collection

Tested in the blood. The plasma collected from blood samples is a dynamic equilibrium relationship between the exposure of the test substance in the plasma and the concentration of the target or toxicity target, and the test substance easily enters the system of animals and humans. When the substance exposure cannot reflect the toxic reaction of the target tissue or organ, it may be necessary to consider using urine, other body fluids, target tissues or organs to determine the concentration of the test substance. The time point of sample collection should try to reach the frequency required for exposure evaluation , But it should not be excessive to avoid disturbing the normal conduct of the test and causing excessive physiological stress response in animals. Normally, in the toxicity test of large animals, the toxicokinetic data is collected from the main research test animal, and the toxicokinetic data of the toxicity test of the trapped animal can be collected from the satellite group test animal for monitoring or characterization. the study.

5. Analysis and determination

Normally, the correlation between the pharmacological effects of the test substance and the concentration of the test substance at the site of action is better than the correlation with the dose. Similarly, the toxic reaction of the test substance has a better correlation with the concentration of the test substance in a specific toxic target organ or tissue. If the test substance is highly permeable at the target site, the concentration of the test substance in the site should be consistent with that in the blood. The test substance is in dynamic balance and a certain ratio, and the concentration of the test substance in the plasma or blood can be measured to reflect the exposure of the test substance at the target site.

When the system exposure of the test substance lacks a good correlation with the toxic reaction, careful analysis should be carried out. Generally, there are two situations:

    ① The selected analyte is incorrect, it is not the material basis of toxicity;

    ②The changes between the systemic exposure of the whole body and the exposure of toxic target organs or organs are not parallel. At this time, it is necessary to measure the exposure of the target site to evaluate its toxicity or metabolic mathematical model to expose the relationship between the systemic exposure and the exposure of the toxic target organ, and use this relationship to indirectly reflect the relationship between systemic exposure and toxicity .

6. Evaluation of the production of anti-drug antibodies

Human-targeted biomacromolecule drugs administered to preclinical laboratory animals, especially after repeated repetitions, may stimulate the immune system to produce anti-drug antibodies (ADA). Subcutaneous injections are earlier than intramuscular and intravenous injections. easy to appear. In short, due to the production of ADA, it will affect the in vivo TK concentration-time characteristics of macromolecular drugs, the degree of exposure, the efficacy and the interpretation of preclinical toxicity data. And to reduce the interference of high concentrations of test substances in plasma or serum on the determination of ADA, it is usually necessary to collect blood samples during the recovery period after the last step of the repeated toxicity test for the determination of ADA.

7. Report

The complete toxicokinetic data should include self-evaluation of the toxicokinetic research results and related explanations of toxic reactions, and report analysis methods to explain the reasons for replacing biophysics and analytes in the analysis. In the result analysis, the safety range of the test substance should be compared and analyzed.

3.5. Application of toxicokinetics in different toxicity experiments

1. Single toxicity test

The toxicokinetic research results of a single toxicity test are helpful for evaluating and predicting the choice of dosage form. After changing the exposure rate and duration, you can also choose the appropriate dosage level in subsequent studies.

2. Repeated toxicity test

The content of toxicokinetic research should generally be carried out in the repeated toxicity test design, which includes the first transition to the periodic exposure monitoring and characteristic research that gradually ends the whole process. The protocol used in subsequent toxicity tests can be revised or adjusted based on the results of the toxicology study. When the early toxicity test has unexplainable toxicity problems, it may be necessary to extend or shorten the time for toxicity monitoring and characteristic research of the test substance, or revise the research content.

3. Genotoxicity test

When the in vivo genotoxicity test result is negative, the exposure data should be combined to assess the genotoxicity risk, especially when the in vitro test shows a clear positive result or the in vitro microbial cell test is not performed.

The assessment of in vivo exposure should use the same animal species, strains and transformation pathways as the genetic toxicity test, at the highest dose or other relevant doses. In vivo exposure can be determined by the in vivo cytotoxicity (such as a significant change in the ratio of immature red blood cells to the total number of red blood cells in the tissue detected in the micronucleus test) or exposure (measurement of the test substance and/or in the blood or plasma). Or the exposure of metabolites, or the direct determination of the test substance in the target tissue and/if the in vitro genotoxicity test results are negative, the above methods or the results of the dentate pharmacokinetics/toxicity test results for other purposes can be used, combined Evaluation is performed in vivo.

4. Reproductive toxicity test

The main purpose of the reproductive toxicity toxicokinetics study is to analyze the results of the reproductive toxicity test, which helps to determine whether the different doses at different stages in the reproductive toxicity test have reached sufficient exposure. The kinetic characteristics of gestational and non-pregnant animals should be considered. The toxicokinetic data should include fetal/larval data to evaluate whether the test substance and/or metabolite can pass through the placental barrier or secretion of milk.

5. Carcinogenicity test

In order to obtain useful toxicokinetic data for the main study, appropriate toxicokinetic monitoring or characterization should be carried out in the dose exploration study, and special attention should be paid to animal species, strains, and first-time responses that were not used in the early toxicity test. According to the possible systemic exposure of the tested animals and humans, the appropriate highest dose in the carcinogenicity test is determined. The systemic exposure of the dose selected in the carcinogenicity test should exceed several times the exposure when the maximum therapeutic dose is used in humans. The main research protocol, the selection of animal species and strains should be based on the existing pharmacokinetics and toxicokinetics.

It is recommended to monitor to ensure that the exposure in the main study is consistent with the kinetic characterization obtained in an independent or specific dose exploration study. This kind of kinetic monitoring can be done at certain time points in the test, more than 6 months. The monitoring is usually unnecessary.

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